SUMMARY Regulation of transcription is largely focused on the initiation process that involves components of the basal transcription machinery (RNA polymerase II and the general transcription factors) and Mediator, a large (25 polypeptides, MW ~1 MDa) multi-subunit complex conserved throughout eukaryotes that is essential for transcriptional response to regulatory information. All of these components come together to form a giant preinitiation complex (over 60 different polypeptides, MW ~2.5 MDa) responsible for transcription initiation. In this application we propose to apply the most advanced macromolecular cryo-electron microscopy (cryo- EM) and image analysis techniques to determine and analyze the structures of key transcription complexes and investigate the mechanism of transcription regulation by Mediator. Cryo-EM image analysis is uniquely suited to provide information about these large and structurally dynamic complexes and our EM studies (along with the work of many others) have contributed significantly to the current understanding of transcription regulation. We now propose to extend this work to obtain structural information that will reveal the mechanism of regulation. In Aim 1 we will take advantage of our recent breakthrough in Mediator purification to obtain a cryo-EM structure of Mediator with high enough resolution to identify density corresponding to individual protein subunits. 3D difference mapping and molecular labeling techniques will be used to localize specific subunits and reveal the overall organization of the Mediator complex. In Aim 2 we will determine how activators and transcription factors influence Mediator conformation in a way that might enable regulation by affecting the interaction of Mediator with RNA polymerase II and the general transcription factors. In Aim 3 we will determine and analyze the 3D structure of a minimal preinitiation complex (a catalytic core comprising RNAPII, TBP, TFIIB, TFIIF, and promoter DNA) in combination with the Head complex, the Mediator module directly responsible for transducing regulatory information to the preinitiation complex. This structure will reveal how Mediator might enable regulation through effects on the assembly, stability, and structure of the preinitiation complex. Completion of these aims will provide structural information critical for unraveling the mechanism of transcription regulation.